ABSTRACTMicroglia actively survey the brain microenvironment and play essential roles in sculpting synaptic connections during brain development. While microglial functions in the adult brain are less clear, activated microglia can closely appose neuronal cell bodies and displace axosomatic presynaptic terminals. Microglia-mediated stripping of presynaptic terminals is considered neuroprotective, but the cellular and molecular mechanisms are poorly defined. Using 3D electron microscopy, we demonstrate that activated microglia displace inhibitory presynaptic terminals from cortical neurons in adult mice. Electrophysiological recordings further establish that the reduction in inhibitory GABAergic synapses increased synchronized firing of cortical neurons in γ-frequency band. Increased neuronal activity results in the calcium-mediated activation of CaM kinase IV, phosphorylation of CREB, increased expression of antiapoptotic and neurotrophic molecules and reduced apoptosis of cortical neurons following injury. These results indicate that activated microglia can protect the adult brain by migrating to inhibitory synapses and displacing them from cortical neurons.

f2: 3D-EM reveals axosomatic synaptic displacement by activated microglia.(a) Electron micrograph of a microglia (‘M’) closely apposing part of a neuronal soma (‘N’) 24 h after the final LPS injection. Arrowheads indicate axosomatic inhibitory synapses. A region of interest is outlined in the box in a and serial sections of this region are shown in b. Scale bar, 1 μm. (b) In these serial EM sections, the leading edge of the microglial process (blue) partially displaces the presynaptic terminal (red), while the remainder of this terminal retains a normal synaptic cleft with the neuronal perikarya (yellow). Pairs of arrowheads indicate close contact between the microglia, the neuron and the synaptic terminal. (c) 3D reconstruction of the serial images shown in b. (d) Representative electron micrographs of motor cortex neuropil from PBS- and LPS-injected mice. Arrows point to asymmetric excitatory synapses. Scale bar, 1 μm. (e) The densities of asymmetrical excitatory synapses were similar in the motor cortices from PBS- and LPS-injected mice. NS, not significant; t-test.

Mentions:
Laminar layers III to V of frontal lobe motor cortices of PBS- or LPS-treated mice were processed and stacks of consecutive electron micrographs were obtained using a Zeiss 3D-scanning electron microscope equipped with a Gatan in-chamber ultramicrotome23. Microglia were identified by their characteristic electron-dense cytoplasm containing conspicuous inclusion bodies, elongated mitochondria and late lysosomes24. A representative image (Fig. 2a) shows an activated microglia (‘M’) closely apposing part of a neuronal soma 24 h after the final LPS injection. Where the microglia is absent, presynaptic boutons with abundant synaptic vesicles are distributed around the neuronal surface membrane containing symmetrical post-synaptic densities, indicative of inhibitory synapses25 (Fig. 2a, arrowheads). Conversely, where the microglia tightly apposes the neuronal perikarya (‘N’), presynaptic structures are not present.

f2: 3D-EM reveals axosomatic synaptic displacement by activated microglia.(a) Electron micrograph of a microglia (‘M’) closely apposing part of a neuronal soma (‘N’) 24 h after the final LPS injection. Arrowheads indicate axosomatic inhibitory synapses. A region of interest is outlined in the box in a and serial sections of this region are shown in b. Scale bar, 1 μm. (b) In these serial EM sections, the leading edge of the microglial process (blue) partially displaces the presynaptic terminal (red), while the remainder of this terminal retains a normal synaptic cleft with the neuronal perikarya (yellow). Pairs of arrowheads indicate close contact between the microglia, the neuron and the synaptic terminal. (c) 3D reconstruction of the serial images shown in b. (d) Representative electron micrographs of motor cortex neuropil from PBS- and LPS-injected mice. Arrows point to asymmetric excitatory synapses. Scale bar, 1 μm. (e) The densities of asymmetrical excitatory synapses were similar in the motor cortices from PBS- and LPS-injected mice. NS, not significant; t-test.

Mentions:
Laminar layers III to V of frontal lobe motor cortices of PBS- or LPS-treated mice were processed and stacks of consecutive electron micrographs were obtained using a Zeiss 3D-scanning electron microscope equipped with a Gatan in-chamber ultramicrotome23. Microglia were identified by their characteristic electron-dense cytoplasm containing conspicuous inclusion bodies, elongated mitochondria and late lysosomes24. A representative image (Fig. 2a) shows an activated microglia (‘M’) closely apposing part of a neuronal soma 24 h after the final LPS injection. Where the microglia is absent, presynaptic boutons with abundant synaptic vesicles are distributed around the neuronal surface membrane containing symmetrical post-synaptic densities, indicative of inhibitory synapses25 (Fig. 2a, arrowheads). Conversely, where the microglia tightly apposes the neuronal perikarya (‘N’), presynaptic structures are not present.

Bottom Line:
Electrophysiological recordings further establish that the reduction in inhibitory GABAergic synapses increased synchronized firing of cortical neurons in γ-frequency band.Increased neuronal activity results in the calcium-mediated activation of CaM kinase IV, phosphorylation of CREB, increased expression of antiapoptotic and neurotrophic molecules and reduced apoptosis of cortical neurons following injury.These results indicate that activated microglia can protect the adult brain by migrating to inhibitory synapses and displacing them from cortical neurons.

ABSTRACTMicroglia actively survey the brain microenvironment and play essential roles in sculpting synaptic connections during brain development. While microglial functions in the adult brain are less clear, activated microglia can closely appose neuronal cell bodies and displace axosomatic presynaptic terminals. Microglia-mediated stripping of presynaptic terminals is considered neuroprotective, but the cellular and molecular mechanisms are poorly defined. Using 3D electron microscopy, we demonstrate that activated microglia displace inhibitory presynaptic terminals from cortical neurons in adult mice. Electrophysiological recordings further establish that the reduction in inhibitory GABAergic synapses increased synchronized firing of cortical neurons in γ-frequency band. Increased neuronal activity results in the calcium-mediated activation of CaM kinase IV, phosphorylation of CREB, increased expression of antiapoptotic and neurotrophic molecules and reduced apoptosis of cortical neurons following injury. These results indicate that activated microglia can protect the adult brain by migrating to inhibitory synapses and displacing them from cortical neurons.